Title Molecular Basis of mRNA Export Abstract One of the great hallmarks of evolution is the enclosure of genetic information in the nucleus. The spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that allow bi-directional macromolecular exchange across the nuclear envelope and thus function as key regulators of the flow of genetic information from DNA to RNA to protein. The NPC is a massive transport channel that is constructed by ~30 distinct proteins, termed nucleoporins (nups), which assemble into six evolutionarily conserved subcomplexes. Due to 8-fold symmetry, each subcomplex is present in multiple copies in the fully assembled NPC, such that the entire assembly reaches the extraordinary molecular mass of ~120 MDa in vertebrates. The NPC coat, a protein shell that directly interacts with the inner and outer membranes of the nuclear envelope, is assembled from multiple copies of the hetero-heptameric coat nucleoporin complex (CNC). The NPC coat anchors the donut-shaped NPC core harboring the central transport channel, and provides key binding sites for asymmetric nucleoporins on its exposed nucleoplasmic and cytoplasmic faces that, in turn, recruit various transcription and mRNA export machineries to the NPC. These associated machineries establish transport directionality and allow the NPC to play a major role in gene regulation. As such, it is unsurprising that genetic modifications of asymmetric nucleoporins have been associated with a diverse set of human conditions, such as neoplastic diseases or susceptibility to escalating viral infections. In higher eukaryotes, the asymmetric cytoplasmic filament nucleoporin Nup358, which has no homolog in single-cell eukaryotes, is required for the efficient synthesis of secretory proteins that depends on an alternative RNA export (ALREX) pathway. ALREX mRNAs are characterized by a signal sequence-coding region (SSCR), which encodes the short hydrophobic consensus polypeptide required for protein targeting to the endoplasmic reticulum. At the cytoplasmic face of the NPC, Nup358 interacts with ALREX mRNAs through direct binding to their SSCR and is required for their efficient translation. In human Nup358, the domain mediating SSCR binding has been identified as a susceptibility locus for a hereditary form of acute necrotizing encephalopathy (ANE), a rare but severe condition developed by some children upon common viral infections. Although the etiology of ANE remains unknown, increased levels of pro-inflammatory cytokines in combination with the shared involvement of Nup358 in the ALREX pathway as well as ANE support the hypothesis that a deregulation of secretory protein expression may play a relevant role. In this proposal, we seek to elucidate the atomic architecture of the cytoplasmic face of the NPC and establish the molecular mechanism of ALREX pathway regulation by Nup358 while assessing its relevance to secretory protein synthesis and ANE. These goals will be achieved through a combination of biochemical, structural and functional approaches designed to dissect molecular interactions between the relevant nucleoporins including wild type and ANE-associated Nup358 variants and SSCR RNA. Overall, our research will contribute to the understanding of fundamental mRNA export mechanisms with particular relevance to the synthesis of the secretory proteome and may provide therapeutically important clues to human diseases associated with mRNA export dysfunction and the development of ANE, a frequently untreatable and ultimately fatal nucleoporin disease.